Hereditary Non-Polyposis Colon Cancer (HNPCC) is a common cancer predisposition syndrome characterized by a dominant mode of transmission and a high penetrance (Lynch et al., Gastroenterology 104:1535-1549 (1993)). Approximately 5% to 15% of colon cancers in industrialized nations have been attributed to HNPCC, suggesting an allele frequency that could be as high as 1 in 200 (Papadopoulos et al., Science 263:1625-1629 (1994); Ponz de Leon et al., Cancer 71:3493-3501 (1993); Kee and Collins, Gut 32:509-512 (1991); Cannon-Albright et al., N. Engl. J. Med. 319:533-537 (1988)). Although colon cancer is the principal cancer associated with HNPCC, more than 35% of the people in these families suffer other types of tumors of which endometrial and ovarian tumors are the most common. (Bishop and Thomas, Cancer Surveys 9:585-604 (1990)).
Genetic linkage studies have identified two HNPCC loci that are thought to account for nearly 90% of the affected families (Peltomaki et al., Science 260:810-812 (1993a); Lindblom et al., Nature Genetics 5:279-282 (1993); Nystrom-Lahti et al., Am. J. Hum. Genet. 55:659-665 (1994)). The first genetic locus characterized, which appears to account for approximately 50-60% of the HNPCC cases, was found to be located on chromosome 2p21 where mutations in the human MutS Homologue (hMSH2) gene were found to cosegregate with the disease (Fishel et al., Cell 75:1027-1038 (1993); Leach et al., Cell 75:1215-1225 (1993)).
The second locus, which appears to account for up to 20-30% of the HNPCC cases, was found to be located on chromosome 3p21 where mutations in the human MutL Homologue (hMLH1) gene were found to cosegregate with the disease (Bronner et al., Nature 368:258-261 (1994); Papadopoulos et al., Science 263:1625-1629 (1994)). These two genes code for homologues of the bacterial MutS and MutL proteins, which are essential components of the post-replication mismatch repair machinery (For a review see: Fishel and Kolodner, Curr. Op. Genet. Devel. 5:382-395 (1995)). Mutation of these genes in bacteria results in a generalized mutator (mut) phenotype that has been attributed to the absence of repair functions capable of recognizing mismatched nucleotides introduced into nascent DNA chains as a result of polymerase misincorporation errors. Such mismatches would subsequently lead to the passive accumulation of spontaneous mutations after a second round of DNA replication. Biochemical studies have shown that the MutS protein is involved in the initial mismatch recognition step and the MutL protein appears to link the excision repair machinery to mismatch recognition (Modrich, Ann. Rev. Genet 25:229-253 (1991)). These results support a direct role for mismatch repair functions in mutation avoidance.
Two other MutL homologues have been described, HPMS1 and hPMS2, that are related to the S. cerevisiae PMS1 gene which was originally identified as a contributor to "Post-Meiotic Segregants": a genetic phenomenon that suggested unrepaired mismatched nucleotides following chromosomal recombination (Nicolaides et al., Nature 371:75-80 (1994)). Mutations of these genes have been found in sporadic colorectal tumors and, in the case of hPMS2, in HNPCC families and may account for an additional 5-10% of HNPCC cases (Parsons et al., Science 268:738-470 (1995)).
The identification of hMSH2 and hMLH1 as genes in which mutations may predispose individuals to HNPCC, was facilitated by the observation that &gt;85% of the tumors derived from these patients displayed instability of simple repetitive (microsatellite) sequences (Aaltonen et al., Science 260:812-816 (1993)). A similar nucleotide repeat instability was first observed in both E. coli and S. cerevisiae only when their respective mismatch repair genes were defective (Levinson and Gutman, Nucleic Acids Research 15:5313-5338 (1987); Strand et al., Nature 365:274-276 (1993)). Microsatellite instability has been observed in 5-85% of a variety of sporadic tumors (often termed: RER+ for replication error positive) suggesting that defects in mismatch repair or some related replication fidelity process may contribute widely to tumorigenesis (Ionov et al., Nature 363:558-561 (1993); Thibodeau et al., Science 260:816-819 (1993); Risinger et al., Cancer Res. 53:5100-5103 (1993); Young et al., Hum. Mutat. 2:351-354 (1993); Han et al., Cancer Res. 53:5087-5089 (1993); Peltomaki et al., Cancer Res. 53:5853-5855 (1993b); Gonzalez-Zulueta et al., Cancer Res. 53:5620-5623 (1993); Rhyu et al., Oncogene 9:29-32 (1994); Wada et al., Blood 83:3449-3456 (1994); Shridhar et al., Cancer Res. 54:2084-2087 (1994); Merlo et al., Cancer Res. 54:2098-2101 (1994); Wooster et al., Nature Genet. 6:152-156 (1994); Yee et al., Cancer Res. 54:1641-1644 (1994); Burks et al., Oncogene 9:1163-1166 (1994); Schoenberg et al., Biochem. Biophys. Res. Commun. 198:74-80 (1994); Honchel et al., Cancer Res. 54:1159-1563 (1994); Shibata et al., Nature Genet. 6:273-281 (1994); and Aaltonen et al., Cancer Res. 54:1645-1648 (1994)). In addition to microsatellite instability, cell lines that contain mutations in hMSH2 or hMLH1 are also defective for mismatch repair in vitro (Umar et al., J. Biol. Chem. 259:1-4 (1994a); Parsons et al., Science 75:1227-1236 (1993)), they display a generalized increase in spontaneous mutation frequency (Bhattacharyya et al., Proc. Natl. Acad. Sci. USA 91:6319-6323 (1994)) and are resistant to alkylating agents (Kat et al., Proc. Natl. Acad. Sci. USA 90:6424-6428 (1993)). The connection of microsatellite instability to a generalized mutator phenotype has suggested that the detection of such changes might be used as a convenient molecular diagnosis of a mismatch repair defect and a mutator phenotype in clinically presented tumors.
In both bacteria and yeast, MutS and its homologues play additional roles in genetic recombination. Mutational studies have shown that recombination between closely spaced markers is increased (Fishel et al., J. Mol. Biol. 188(2):147-157 (1986); Feinstein and Low, Genetics 113:13-33 (1986); Jones et al., Cell 50:621-626 (1987)) and the length of DNA tracts exchanged between recombining chromosomes is reduced (Alani et al., Genetics 137:19-39 (1994)) in MutS (or MSH2) deficient cells. In addition, the tolerance of heterologous DNA sequence in recombining chromosomes is substantially increased in such bacterial or yeast cells (Rayssiguier et al., Nature 342(6248):396-401 (1989); Selva et al., Genetics 139:1175-1188 (1995)). These results suggest that mismatched nucleotides formed during genetic recombination provide a target for mismatch repair functions which, in the case of multiple mismatches, results in abortion of the recombination process (Radman, Genome 31(1):68-73 (1989); and Worth et al., Proc. Nat. Acad. Sci. USA 91:3238-3241 (1994)). This later observation has particular relevance to carcinogenesis since large scale rearrangements between non-homologous and/or partially homologous chromosomal sequences are a hallmark of tumor cells and may be indicative of widespread reduced-fidelity recombination processes.
Although the precise function of hMLH1 is poorly understood, hMSH2 has been purified and found to bind insertion/deletion loop-type (IDL) mismatched nucleotides with high affinity, and the single base pair G/T mismatch with lower affinity (Fishel et al., Science 266:1403-1405 (1994a); Fishel et al., Cancer Res. 54:5539-5542 (1994b)). IDL mismatched nucleotides have been proposed as an intermediate in microsatellite instability (Kunkel, Nature 365:207-208 (1993)). Furthermore, biochemical reconstitution studies that examine mismatch repair functions in vitro (Umar et al., J. Biol. Chem. 259:1-4 (1994a); Umar et al., Science 266:814-816 (1994b)), have resulted in the purification of two activities that appear to complement extracts derived from cell lines with known mutations in hMSH2 and hMLH1. Interestingly, both these purified complementing fractions consist of tightly complexed heterodimers. Protein extracts of the LoVo cell line, which contains deletions of both hMSH2 alleles, are complemented by a heterodimer that consists of hMSH2 (105 kDa) and a 160 kDa protein (P. Modrich, Ann. Rev. Genet. 25:229-253 (1991)), that has been cloned and identified as another MutS homologue a GT binding protein (GTBP/P160) and which has been found to co-purify as a heterodimer with hMSH2). There is some suggestion that the hMSH2/GTBP/160 heterodimer may bind mismatched nucleotides with a much higher affinity than either of the corresponding individual proteins. Protein extracts of the HCT116 cell line, which contains a deletion and a non-sense mutation of the hMLH1 alleles, are complemented by a heterodimer of hMLH1 (84 kDa) and hPMS2 (110 kDa) (Li and Modrich, Proc. Nat. Acad. Sci. USA 92:1950-1954 (1995)).
There are multiple possibilities by which faulty mismatch repair genes could result in the development of cancer (Fishel and Kolodner, Curr. Op. Genet. Devel. 5:382-395 (1995)). It has been hypothesized that cells with repair defects might have elevated rates of mutations (Loeb, Cancer Res. 51:3075-3079 (1991)). The accumulation of mutations could result in growth control defects--such as by interfering with check-point control mechanisms, tumor suppressors, or oncogenes that cause mutant cells to progress to malignancy. It is clear that an appropriate animal model is needed to investigate the possible role(s) of mismatch repair in tumorigenesis and to provide systems for testing of therapeutic interventions for the treatment of cancer and other diseases associated with mismatch repair deficiencies.
Although mutations in the human MSH2 gene co-segregate with malignant disease in a number of HNPCC kindreds, it has remained debatable whether mismatch repair is involved directly in the onset of tumorigenesis. Described below are mice having one or more disrupted MSH2 alleles. These mice are useful for the study of the role of mismatch repair in oncogenesis and as screening tools for suspected charcinogens and chemotherapeutic agents. While these mice are fertile and viable through at least 3 generations, they succumb to tumors at an early age with high frequency, supporting a role for MSH2 in tumorigenesis.